Proc. Nati. Acad. Sci. USA Vol. 86, pp. 7186-7190, September 1989 Medical Sciences Loss of distinct regions on the short arm of 17 associated with tumorigenesis of human astrocytomas (tumor suppressor /antioncogenes/brain tumors/neurofibromatosis/colon cancer) M. EL-AzOUZI*, R. Y. CHUNG*, G. E. FARMER*, R. L. MARTUZA*, P. McL. BLACKt, G. A. ROULEAUt, C. HETTLICH*, E. T. HEDLEY-WHYTE§, N. T. ZERVAS*, K. PANAGOPOULOS*, Y. NAKAMURA¶, J. F. GUSELLAt, AND B. R. SEIZINGER*tII *Molecular Neurooncology Laboratory, Neurosurgery Service, tMolecular Neurogenetics Laboratory, and §Neuropathology Laboratory, Massachusetts General Hospital, and Harvard Medical School, Boston, MA 02114; *Department of Neurosurgery, Brigham and Women's Hospital, and Harvard Medical School, Boston, MA 02115; and lHoward Hughes Medical Institute, and University of Utah, Salt Lake City, UT 84132 Communicated by Richard L. Sidman, June 28, 1989 (received for review February 2, 1989)

ABSTRACT Astrocytomas, including glioblastoma multi- differentiated astrocytomas and the glioblastoma multiforme. forme, represent the most frequent and deadly primary neo- Although some patients with anaplastic astrocytoma respond plasms of the human nervous system. Despite a number of well to chemotherapy and/or radiotherapy, other patients do previous cytogenetic and oncogene studies primarily focusing not (2). Anaplastic astrocytomas, therefore, may be com- on malignant astrocytomas, the primary mechanism of tumor posed of several distinct biological subgroups, which cannot initiation has remained obscure. The loss or inactivation of be detected by standard histopathological techniques (6, 7). "tumor suppressor" genes are thought to play a fundamental Thus, alternative diagnostic tools, such as genetic markers, role in the development ofmany human cancers. Thus, we have could be useful in further subclassifying glial tumors and analyzed astrocytomas of various histological malignancy leading to more accurate grades with polymorphic DNA markers to search for specific clinical prognosis and more appro- chromosomal deletions potentially pointing to loci containing priate choice of therapies. tumor suppressor genes. Loss of constitutional heterozygosity In the present study, we have analyzed astrocytomas of indicating chromosomal loss or deletions was most frequently various malignancy grades with polymorphic DNA markers seen for markers on the short arm of in 50% to search for the loss of chromosomal regions containing of the informative tumors (5 of 10 informative cases) and, to a tumor suppressor genes (for review, see ref. 8). This tech- lesser extent, for markers on 1 and 10. Deletions nique is particularly powerful for uncovering chromosomal on chromosome 17p were seen in both low-grade and high- deletions in benign solid tumors (9-11); karyotyping in these grade manant astrocytomas, suggesting that this chromo- tumors is often unsuccessful due to their low mitotic index. some may contain a tumor suppressor associated with the Moreover, molecular probes may detect small (interstitial) early events in tumorigenesis. The common region of deletions deletions and mitotic recombinations not detectable by stan- on the short arm of chromosome 17 is, therefore, dearly dard cytogenetic techniques. Markers for chromosomes 17 distinct from the gene causing von Recklinghausen neurofibro- and 22 were chosen because these chromosomes contain the matosis (NF1), a tumor syndrome associated with glial tumors two types of neurofibromatosis, NF1 and NF2, respectively that maps to the long arm of chromosome 17. The search for (9-14). Both forms of neurofibromatosis are associated with progressively smaller deletions on chromosome 17p in astro- certain types ofglial tumors (for review, see refs. 15 and 16). cytomas may be the way to clone and characterize this locus, Chromosomes 1, 7, and 10 were selected because ofprevious thus leading to insights into normal and abnormal growth and reports on their frequent numerical and structural aberrations differentiation of gial cells. in glial tumors (17-21).

Gliomas are the most common primary tumors of the human MATERIALS AND METHODS central nervous system (1). The majority of brain tumors are astrocytomas (1), which are most commonly divided into DNA of high relative molecular weight was isolated from 14 three histopathological malignancy groups (2-4). However, astrocytomas and corresponding normal tissues (peripheral alternative classification systems are in use, including the leukocytes) as described (9, 10). Tumors A3, A6, A13, and classical four-tiered system by Kernohan (2-4), and even A14 are recurrent tumors; tumors A3 and A6 were irradiated experienced neuropathologists may disagree in their assign- before the tumor specimens analyzed in this study were ments of astrocytomas to a precise malignancy grade. Ac- removed. All other specimens represent primary tumors cording to the three-tiered classification, the first and least removed before any chemotherapy or radiotherapy. Tumor malignant type is the well-differentiated astrocytoma (astro- A14 was from a patient with NF1; all other cases represent cytoma grade I). These tumors are often manageable by sporadic tumors from patients without any history of NF1 or surgery, although recurrence is frequent. The recurrent tu- any other hereditary tumor syndrome. mors are often less differentiated and of higher grade, sug- Representative samples of each tumor were histopatho- gesting that they are derived from the less malignant forms in logically analyzed by two experienced neuropathologists at a progressive fashion. Glioblastoma multiforme (astrocy- the Massachusetts General Hospital, using a three-tiered toma grade III) is the most malignant type and is virtually classification system of malignancy, according to that estab- always lethal, despite the best available surgery, radiation, lished by the World Health Organization, modified by Zulch and chemotherapy (5). The anaplastic astrocytomas (astro- (4) (Table 1). cytoma grade II) are intermediate between the well- Abbreviations: NF1 and -2, neurofibromatosis type 1 and 2, respec- The publication costs of this article were defrayed in part by page charge tively. payment. This article must therefore be hereby marked "advertisement" 'To whom correspondence and reprint requests should be ad- in accordance with 18 U.S.C. §1734 solely to indicate this fact. dressed. 7186 Downloaded by guest on September 29, 2021 Medical Sciences: El-Azouzi et al. Proc. Natl. Acad. Sci. USA 86 (1989) 7187

Table 1. Loss of heterozygosity for loci on chromosome 17p in astrocytomas Chromosome (probe and RFLP enzyme) 1 2 3 7 10 10 10 10 (pYNZ2 (pYNH24 (H3H2 (pJCZ67 (pEFD75 (5-1 (OAT (OAT Patient Tumor type Msp I) Msp I) HindIII) Rsa I) Taq I) Taq I) Msp I) Rsa I) Al Astro II 12 12 12 A2 Astro I-II* 12 12 A3 Astro III 12 12 12 A4 Astro III 12 A5 Astro II 2 12 12 A6 Astro III 2 12 12 12 12 12 A7 Astro I-II* 12 12 12 12 A8 Astro I-II* 12 12 A9 Astro I 12 A10 Astro III 12 12 12 12 12 All Astro II-III* 12 A12 Astro III 12 A13 Astro I 12 12 12 A14 Astro III 12 1 Chromosome (probe and RFLP enzyme) 17 17 17 17 17 17 17 17 17 22 22 22 (pYNZ22 (pYNH37.3 (pHF12-1 (EW301 (EW203 (EW203 (EW206 (EW207 (pTHH59 (p22/34 (pMS3-18 (W13E Patient Rsa I) Taq I) Msp I) Bgl II) Bgl II) Taq I) Msp I) HindIII) Taq I) Taq I) Bgl II) Taq I) Al 12 12 12 12 A2 2 12 - - 12 12 A3 12 12 12 12 A4 12 A5 12 - 12 - 12 12 A6 12 12 - 12 12 12 A7 2 1 12 12 12 12 A8 12 - 12 12 12 12 12 12 12 A9 12 12 12 AlO 2 2 12 12 12 12 12 12 All A12 2 1 2 2 12 A13 - - 12 12 12 12 A14 2 1 2 Tumor DNA and normal DNA from astrocytoma patients was analyzed with polymorphic DNA markers for loss of constitutional heterozygosity as described. Phenotype of the tumor tissue is shown for every case where blood DNA displayed heterozygosity. 12, Heterozygosity (even though different allele pairs may be present for certain multiallele markers); 1, continued presence of the larger allelic restriction fragment and loss ofthe smaller allelic fragment relative to normal tissue DNA; 2, continued presence ofthe smaller allelic restriction fragment and loss of the larger fragment. A minus sign indicates that normal DNA was tested but proved uninformative because the DNA did not display heterozygosity. Absence ofan entry indicates that a marker was not tested or did not give a readable result for that particular patient. All astrocytomas were independently graded by the two neuropathologists. Tumors A2, A7, A8, and All (marked with an asterisk) were classified by one neuropathologist as grades I, I, I, and III, respectively; the other neuropathologist classified all four tumors as grades II. However, both agreed that tumors A2, A7, and A8 represent low-grade astrocytomas, whereas All is of higher-grade malignancy. For probe definitions, see Materials and Methods. Astro, astrocytoma. Approximately 5 ,g of normal and tumor DNA were gration. To determine whether loss of one allele for chromo- digested to completion with an appropriate restriction en- some 17 in the tumor tissue was associated with duplication zyme, fractionated by agarose gel electrophoresis, trans- of the remaining allele, the hybridization signals for chromo- ferred to nylon membrane, and hybridized to 32P-labeled some 17 probes were normalized to those obtained when the probe DNA (9, 10). The following probes known to reveal same Southern blots were rehybridized with probes for loci restriction fragment length polymorphism in human genomic on other chromosomes (9-11). DNA for loci on several different chromosomes were used: pYNZ2 (D17S57) (22), pYNH24 (D2S44) (23), H3H2 (DNF15S2) (24), pJCZ67 (D7S396) (25), pEDF75 (D10S25) RESULTS (26), 5-1 (D1OS1) (26), ornithine aminotransferase (OAT) (26), pYNZ22 (D17S30) (27), pYNH37.3 (D17S28) (27), pHF12-1 Loss of constitutional heterozygosity pointing to chromo- (D17S1) (27), EW301 (D17S58) (27), EW203 (D17S54) (27), somal loss or deletions was seen for loci on chromosome 1 in EW206 (D17S57) (27), EW207 (D17S73) (27), pTHH59 2 of 6 informative cases, on chromosome 10 in 1 of 7 (D17S4) (27), pMS3-18 (D22S1) (28), p22/34 (D22S9) (28), informative cases, and on chromosome 17 in 5 of 13 infor- and W13E (D22S21) (28) [see Human Gene Mapping 9.5 mative cases (Table 1). Ten of the 13 tumors informative for (1988): Update to the Ninth International Workshop on any marker on chromosome 17 (including long arm and short Human Gene Mapping (22-28)]. arm) were informative for markers on the short arm of this Autoradiograms were analyzed by scanning densitometry chromosome. Five of these 10 tumors (50%o) lost constitu- with a LKB Ultrascan XL. The peak areas corresponding to tional heterozygosity (Fig. 1). No loss of heterozygosity was each hybridization signal were calculated by electronic inte- seen for loci on chromosome 2 (13 informative cases), Downloaded by guest on September 29, 2021 7188 Medical Sciences: El-Azouzi et al. Proc. NatL Acad. Sci. USA 86 (1989) Patient pYNZ22 pYNH373 pHF 12.1 EW301

N T N T 13 A2 I 12

N 101 EW203 49 N T N T N T 11.2

EW207 17q N T N T

I- foO FIG. 2. Common region of deletions on the short arm of chro- 0 A14 2- - mosome 17 in astrocytomas. Solid lines at right indicate the deleted 2-4w regions. Broken lines indicate that the tumors may be deleted for these regions, but the respective markers were not informative. Absence ofa line indicates that the tumors were not deleted for these chromosome 17p 17q a _ regions. Tumors A10 and A12 lost heterozygosity for markers on the short arm but maintained heterozygosity for markers on the long arm centromere ofchromosome 17 (EW203 and pTHH59, respectively). Because the on FIG. 1. Specific loss ofheterozygosity at loci on the short arm of likelihood that the centromere was lost together with regions the chromosome 17 in astrocytoma tumor tissue. DNA from tumor and short arm, but without the long arm, is extremely low, the break- points in these two tumors are assumed to be distal to the centromere control tissue (peripheral leukocytes) was analyzed with polymor- on the the phic DNA markers as described. Numbers at left indicate the short arm of chromosome 17 (for localization of markers observed and 2 to the and smaller restriction see ref. 27). Note, however, that only tumors A2 and A14 are alleles, 1 referring larger associated with or whereas fragments, respectively (even though different allele pairs may be "true" chromosomal loss deletions, present in different individuals for certain multiallele markers, in- losses of heterozygosity in tumors A7, A10, and A12 represent cluding pYNZ22, pYNH37.3, and pTHH59). N, DNA from normal mitotic recombinations. NF1 indicates the location of the defective tissue (peripheral leukocytes); T, astrocytoma tumor tissue. gene for von Recklinghausen neurofibromatosis (NF1). and pYNH24 on chromosome 2) assured that they were not chromosome 3 (2 informative cases), chromosome 7 (7 in- detected. Table 2 shows that the ratio of the copy number of formative cases), and chromosome 22 (8 informative cases). chromosome 17p of tumor compared with normal tissue was Loss of heterozygosity on chromosome 17p in 50% of the -1:2 for tumors A2 and A14, suggesting that these two informative tumors represents the most frequent genetic tumors are associated with "true" loss of chromosomal abnormality detected in this study. (The number of losses regions. However, this ratio was :1: 1 in tumors A7, A10, involving chromosomes 1 and 10 is clearly too small to draw and A12, suggesting that loss of heterozygosity in these any significant conclusions.) Loss ofheterozygosity on chro- tumors reflects mitotic recombinations. mosome 17 was not only seen in high-grade astrocytomas (AlO, A12, and A14), but also in low-grade tumors (A2 and A7). DISCUSSION The availability of a number of markers for different Astroctyomas may represent a model system in which to regions on chromosome 17 allowed further bracketing of the study tumor initiation, progression, and malignancy. We common region of deletion. Fig. 2 shows that four of the five have used a molecular genetic approach to show that both tumors that lost heterozygosity for markers on chromosome low- and high-malignancy-grade astrocytomas frequently dis- 17 were associated with partial deletions of the short arm of play loss of distinct regions on chromosome 17p. Thus, this chromosome 17, with the common region ofdeletion mapping region may contain a tumor suppressor gene (or genes) the- between the marker EW301 on the proximal short arm of loss, deletion, or inactivation of which is associated with chromosome 17 and the telomere. tumor initiation or early tumor progression in astrocytomas. To distinguish among different mitotic mechanisms that Four ofthe five tumors with loss ofheterozygosity for loci on might have produced loss of heterozygosity on chromosome chromosome 17 selectively lost regions on the short arm. The 17, the hybridization signals from Fig. 1 were compared with location ofthis putative astrocytoma tumor suppressor locus several probes for control loci on other chromosomes using is, therefore, clearly distinct from that ofthe gene for NF1 on quantitative densitometry. Maintenance of heterozygosity in the proximal long arm of chromosome 17 (29, 30). Because the tumors for these control loci (pYNZ2 on chromosome 1 certain types of glial tumors, including optic gliomas, are Downloaded by guest on September 29, 2021 Medical Sciences: El-Azouzi et al. Proc. NatL. Acad. Sci. USA 86 (1989) 7189 Table 2. Quantitative densitometry of probe hybridization for chromosome 17p were associated with cytogenetically un- astrocytomas with loss of heterozygosity on chromosome 17p detectable mitotic recombinations. This result may explain Chromosome 17p/ Tumor/ the failure ofprevious karyotype studies to recognize genetic Patient control chromosome normal aberrations on chromosome 17p as a frequent event in glial tumors. A2 0.53 The exact role of the putative tumor suppressor locus on Astrocytoma 0.32 chromosome 17p in tumorigenesis of astrocytomas, never- Leukocyte 0.61 theless, is still speculative. The frequent loss of certain A7 1.05 regions of chromosome 17 in both high- and low-grade Astrocytoma 1.19 astrocytomas does not necessarily imply that this genetic Leukocyte 1.13 event represents the primary mechanism of tumor initiation. A10 0.99 Instead, this loss may be involved in tumor maintenance Astrocytoma 0.86 and/or (early) tumor progression, unless genetic linkage Leukocyte 0.86 studies in families displaying this particular tumor type map A12 1.02 the gene to the same chromosomal region. Although pedi- Astrocytoma 1.34 grees with familial gliomas have been reported, the small Leukocyte 1.32 number of living affected individuals in these families will A14 0.47 make it difficult to use the linkage-analysis method to confirm Astrocytoma 0.23 a deleted chromosomal region as the site of the primary Leukocyte 0.49 genetic defect. Furthermore, not all tumor suppressor loci Southern blots hybridized to probes for chromosome 17p (see Fig. may follow the retinoblastoma model of a recessive mecha- 1) were freed from these probes in distilled water for 2 hr at 650C and nism of tumorigenesis, in which loss of both copies of the rehybridized with probes for polymorphic loci on other chromo- gene is required to induce tumor formation (8, 35, 36). somes (control chromosomes). Heterozygosity for restriction frag- Alternatively, the loss of one copy of a tumor suppressor ment length polymorphism (RFLP) at these control loci clearly locus may be sufficient to contribute to tumor maintenance or indicated that these loci were not deleted in the tumor DNA (data not progression. shown). Hybridization signals on the autoradiograms were analyzed by quantitative densitometry; those signals specific to chromosome Although loss ofregions on chromosome 17p is clearly one 17p were normalized to hybridization signals for control chromo- of the more common somatic aberrations, it is obviously not some probes in the same sample. Ratio of the normalized values for the only genetic event in astrocytomas. A confusing hetero- each tumor/normal tissue pair was then calculated. The reliability of geneity of genetic aberrations has been reported mostly for this method has been described (9-11). higher malignancy glial tumors, including amplifications, rearrangements, and overexpressions of various oncogenes associated with NF1 (15), chromosome 17 may contain two (e.g., epidermal growth factor receptor), as well as multiple genes related to tumorigenesis ofglial cells. Curiously, tumor numerical aberrations involving chromosomes 1, 6, 7, 9, 10, A14, an astrocytoma grade III from a patient with NF1 lost 19, 22, and the gonosomes (for review, see ref. 37). A recent the entire copy of chromosome 17, including the NF1 locus molecular genetic study by James et al. (21) suggests that on the long arm, and the putative "astrocytoma locus" on the most glial tumors histologically classified as glioblastoma short arm. However, whether the combined loss of both loci multiforme, but none of the lower-grade astrocytomas, lost on chromosome 17 is of any significance remains to be chromosome 10, consistent with a role of this genetic event determined. in later-stage tumor progression and/or malignancy. Al- p53, a nuclear coded by a gene that maps to the though our study detected no loss of chromosome 10 as a short arm of chromosome 17 (27), was originally believed to particularly frequent event, we note that the number of be an oncogene product. However, recent studies indicate informative tumors was too small to draw any significant that it may actually be the protein product of a tumor conclusions for this chromosome. The same study by James suppressor gene or "antioncogene", similar to the retino- et al. (21) also reports loss of chromosomes 13, 17, and 22 in blastoma gene (for review, see ref. 31). Vogelstein and both low-grade and high-grade gliomas at nonrandom fre- colleagues (32) have recently shown that >75% of colon quencies (14%, 22%, and 19%, respectively) but does not carcinomas are associated with deletions on chromosome further narrow the deleted regions to certain parts of these 17p, including the region 17pl2 to 17pl3.3 that contains the chromosomes. gene coding for p53. Sequence analysis of the coding region Thus, the formation of higher-grade astrocytomas, partic- of the remaining allele for p53 was done in two colon ularly of glioblastoma multiforme, may involve multiple carcinomas and revealed point mutations in a highly con- genetic "hits." Perhaps loss of an astrocytoma tumor sup- served region of the p53-encoding gene in both cases (32). pressor gene on chromosome 17p, at least for some tumors, Thus, the gene encoding p53 may represent a recessive tumor may operate at the primary level of tumor initiation. Addi- suppressor gene important for tumorigenesis of colon carci- tional independent genetic events, such as loss ofother tumor noma. Although no associations are known between the suppressor genes (e.g., on chromosome 10) and/or overex- occurrence of colon cancer and gliomas in the general pop- pression or amplification of oncogenes (e.g., epidermal ulation, multiple families with Turcot syndrome have been growth factor receptor), may lead to a dedifferentiation of reported. In Turcot syndrome, familial polyposis is associ- lower-grade tumors into more malignant forms. According to ated with malignant astrocytomas or medulloblastomas (33, this model, alternative genetic events could eventuate in the 34). Thus, the p53-encoding gene may not only be associated same tumor phenotype. with tumorigenesis of colon carcinoma, but may also be the A similar model oftumorigenesis has been proposed for the target of chromosome 17p deletions in astrocytomas. development of colon carcinoma, including loci on chromo- Somatic loss of constitutional heterozygosity can be somes 5, 17, 18, and 22 (for review, see ref. 8). The loss of a achieved by different alternative mitotic events, including gene on the short arm of chromosome 17 (probably that "true" loss of chromosomal regions and mitotic recombina- coding for p53, see above) is thought to be associated with a tion between the two homologous chromosomes. Both chro- later stage of tumor progression and/or malignancy in colon mosomal mechanisms may unmask a recessive mutant allele carcinoma (32, 38, 39). In contrast, loss of heterozygosity on by elimination of the balancing wild-type gene. Three of the chromosome 17p was seen in low- and high-grade astrocy- S tumors that lost constitutional heterozygosity for loci on tomas, suggesting that it is involved in an earlier stage of Downloaded by guest on September 29, 2021 7190 Medical Sciences: El-Azouzi et al. Proc. NatL Acad. Sci. USA 86 (1989) tumor formation. However, this hypothesis does not neces- 14. Rouleau, G. A., Wertelecki, W., Haines, J. L., Hobbs, W. J., sarily imply that the colon carcinoma gene is different from Trofatter, J. A., Seizinger, B. R., Martuza, R. L., Superneau, the astrocytoma locus. Mutations in the p53 gene may well be D. W., Conneally, P. M. & Gusella, J. F. (1987) Nature (Lon- involved in both tumor types. Such a scenario, in which a don) 329, 246-248. 15. Riccardi, V. M. (1981) N. Engl. J. Med. 305, 1617-1627. tumor suppressor locus is associated with different stages of 16. Martuza, R. L. & Eldridge, R. (1988) N. Engl. J. Med. 318, tumorigenesis in seemingly unrelated tumor types, has been 684-688. suggested for the retinoblastoma gene. Loss or inactivation of 17. Yamada, K., Kondo, T., Yoshioka, M. & Oami, H. (1980) this gene has been implicated in the initiation of retinoblas- Cancer Genet. Cytogenet. 2, 293-307. toma (35, 36, 40, 41) and may have a yet unspecified role in 18. Bigner, S. H., Mark, J., Mahaley, M. S. & Bigner, D. D. (1984) the progression of small cell lung cancer and breast cancer Hereditas 101, 103-113. (42, 43). 19. Rey, J. A., Bello, M. J., de Campos, J. M., Kusak, M. E., The characterization of the Rb gene, the p53 gene, and Ramos, C. & Benitez, J. (1987) Cancer Genet. Cytogenet. 29, other tumor suppressor genes should clarify the pathogenetic 201-221. mechanisms underlying tumor formation with potentially 20. Bigner, S. H., Mark, J., Burger, P. C., Mahaley, M. S., Bull- for and treatment ard, D. E., Muhlbaier, L. H. & Bigner, D. D. (1988) Cancer important implications diagnosis (gene Res. 48, 405-411. replacement therapy) of these common cancers. Study ofthe 21. James, C. D., Carlbom, E., Dumanski, J. P., Hansen, M., normal function of tumor suppressor genes may provide Nordenskjold, M., Collins, V. P. & Cavenee, W. K. (1988) another window on fundamental mechanisms regulating the Cancer Res. 48, 5546-5551. normal development and differentiation of organ systems, 22. Sherman, S. 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